Zataria multiflora Boiss. is used in Iranian traditional medicine to treat gastrointestinal disorders and menrrhalgia. The inhibitory effect of this herb on rat ileum contractions is also reported. The aim of this study was to investigate the effect of Zataria multiflora Boiss. hydroalcoholic leaf extract (ZHLE) on isolated rat uterus in presence of some known uterus stimulants. Pieces of virgin adult rat uterus were mounted in an organ bath containing Tyrode or De Jalon solutions. Uterus contraction was induced by KCl, oxytocin and BaCl2 in presence and absence of certain concentrations of ZHLE. In the oxytocin studies, animals received an injection of oestradiol valerate (5mg/kg, S.C.) 24h prior to the experiment. ZHLE (0.125, 0.25, 0.5, 1 and 2 mg/ml) relaxed the uterus precontracted by KCl (60mM) in a dose-dependent manner (P<0.0001) and at 2mg/ml attenuated the BaCl2 (4mM)-induced uterus contraction (P<0.001). The inhibitory effect of ZHLE on KCl-induced uterus contraction was unaffected by propranolol (1?M). In normal De Jalon solution, ZHLE (0.125, 0.25, 0.5, and 1mg/ml) reduced the oxytocin (10mU/ml)-induced contraction dose-dependently (P<0.0001) but in Ca2+-free De Jalon solution, the stimulatory effect of oxytocin was lesser nevertheless, the inhibitory effect of ZHLE was higher. In presence of atropine (0.5?M), acetylcholine (0.5?M) was failed to induce contraction but KCl (30mM) evoked contraction and the extract diminished the contractile response of KCl. The spasmolytic effect of extract (2mg/ml) on KCl-induced contraction was unaffected by naloxone (1?M). From the obtained results it may be concluded that, the ZHLE may induce the inhibitory effect through blockage of the voltage dependent calcium channels and releasing calcium from intracellular stores in rat uterus smooth muscle. The ineffectiveness of propranolol and naloxone on ZHLE inhibitory effect indicates that adrenergic and opioid agonist substance(s) does not exist in the extract. It seems that there is no anticholinergic substance(s) in the extract. The results support the usage of this plant in traditional medicine.

Copyright ? 2005 by School of PharmacyShaheed Beheshti University of Medical Sciences and Health Services

Original Article

Antispasmodic Effect of Zataria multiflora
Boiss. Leaf Extract

on the Rat Uterus

Mohammad Kazem Gharib Naseri*,
Hamideh Mazlomi, Maryam Goshaiesh,

Gelareh Vakilzadeh and Akbar Heidari

Department of Physiology, School of
Medicine, Jondishapur University of Medical Sciences, Ahwaz, Iran.

Abstract

Zataria multiflora
Boiss. is used in Iranian traditional medicine to treat gastrointestinal
disorders and menrrhalgia. The inhibitory effect of this herb on rat ileum
contractions is also reported. The aim of this study was to investigate the
effect of Zataria multiflora Boiss. hydroalcoholic leaf extract (ZHLE)
on isolated rat uterus in presence of some known uterus stimulants. Pieces of
virgin adult rat uterus were mounted in an organ bath containing Tyrode or De
Jalon solutions. Uterus contraction was induced by KCl, oxytocin and BaCl2
in presence and absence of certain concentrations of ZHLE. In the oxytocin
studies, animals received an injection of oestradiol valerate (5mg/kg, S.C.)
24h prior to the experiment. ZHLE (0.125, 0.25, 0.5, 1 and 2 mg/ml) relaxed the
uterus precontracted by KCl (60mM) in a dose-dependent manner (P<0.0001) and
at 2mg/ml attenuated the BaCl2 (4mM)-induced uterus contraction
(P<0.001). The inhibitory effect of ZHLE on KCl-induced uterus contraction
was unaffected by propranolol (1μM).
In normal De Jalon solution, ZHLE (0.125, 0.25, 0.5, and 1mg/ml) reduced the
oxytocin (10mU/ml)-induced contraction dose-dependently (P<0.0001) but in Ca2+-free
De Jalon solution, the stimulatory effect of oxytocin was lesser nevertheless,
the inhibitory effect of ZHLE was higher. In presence of atropine (0.5μM), acetylcholine (0.5μM)
was failed to induce contraction but KCl (30mM) evoked contraction and the
extract diminished the contractile response of KCl. The spasmolytic effect of
extract (2mg/ml) on KCl-induced contraction was unaffected by naloxone (1μM). From the obtained results it may be concluded that,
the ZHLE may induce the inhibitory effect through blockage of the voltage
dependent calcium channels and releasing calcium from intracellular stores in
rat uterus smooth muscle. The ineffectiveness of propranolol and naloxone on
ZHLE inhibitory effect indicates that adrenergic and opioid agonist
substance(s) does not exist in the extract. It seems that there is no
anticholinergic substance(s) in the extract. The results support the usage of
this plant in traditional medicine.

Keywords:Zataria multiflora
Boiss.; Spasmolytic effect; Rat; Uterus.

Introduction

Zataria multiflora Boiss. from Labiatae grows in Pakistan,
Afghanistan and Iran (1). Different parts of this herb has the same medicinal
effect as origanum and traditionally used for convulsian, relief of
menstruation pain (2) and its antinociceptive and anti-inflammatory effects has
already been reported(1). It?s reported that the antinociceptive effect is
antagonized by naloxone (1). some antifungal (4-6) and antimicrobial effects
have also been? reported (7) for this herb. The main constituents of Zataria
multiflora are carvacrol, thymol, linalol and p-cymene (7). It has
been shown that the hydroalcoholic extract of this herb reduces the
acetylcholine and KCl induced ileum contractions in rat (8). These results
suggested that the voltage dependent calcium channels (VDCCs) are probably
influenced by the herb extract. Since one of the major uses of this herb is to
relief dysmenorrhea, therefore, the present study was designed to investigate
the effect of Zataria multiflora Boiss. leaves hydroalcoholic extract on
isolated rat virgin uterus and possible mechanism(s) of its action.

Experimental

Extract?
preparation

The herb was purchased from local herbal shop in
Ahwaz City (Khuzestan province) and identified by Dr. Naanaie from Khuzestan
Agriculture and Natural Resources Research Center. The leaves were cleaned,
powdered by electric blender and the powder was extracted with 70% alcohol for
72 hours using macerated method. The mixer was filtered with Whatman No 1
filter paper. The solvent of filtrate was evaporated at ambient temperature and
extract powder (13.1% of leaf powder) was kept at 4?C until used.

Animals
and tissue preparation

Adult virgin
female rats (Sprague Dawley) weighting 185 to 225g were kept in Research Center
& Experimental Animal House of Ahwaz University of Medical Sciences under
standard conditions (12/12 light-dark cycle, 20-24?C) with free access to food
and water. The procedure followed was in accordance with Committee of Ethics in
research of Ahwaz University of Medical Sciences. The animals were killed by a
blow on the head, after laparatomy, a piece of uterus (1.5cm) was excised and
mounted in an organ bath (10ml) containing Tyrode?s or De Jalon solutions
(29?C) between two hooks. The lower hook was fixed at the bottom of the organ
bath and the upper hook was connected to an isometric transducer (UF1 Harvard
transducer, UK). The equilibrium period was 60 min in which, the bath solution
was exchanged every 15min. Under 0.5g resting tension, a pen recording system
(Harvard Universal Oscillograph, UK) recorded the uterus contractions. The
composition of Tyrode?s solution (9) was NaCl (137 mM), KCl (2.68 mM), CaCl2
(1.8 mM), NaHCO3 (11.9 mM), MgCl2 (1.05 mM), and NaH2PO4
(0.42 mM) and glucose (5.55 mM). The De Jalon solution was used for oxytocin
experiments (10) with following composition: NaCl (154 mM), KCl (5.6 mM), CaCl2
(0.3 mM), NaHCO3 (1.7 mM), MgCl2 (1.4 mM), and glucose
(5.5 mM). Because of two reasons De Jalon solution was used in oxytocin
experiments: a) the uterus contraction induced by oxytocin in the De Jalon
solution was more stable than in Tyrode solution and b) to prevent
superimposing the extract inhibitory effect on the rhytmic contractions induced
by oxytocin. In the oxytocin protocols, the rats were pretreated with estradiol
valerate (5mg/kg, S.C.) 24h prior to the experiment (10). The salts were
purchased from Merck (Germany), acetylcholine, propranolol, atropine from
Sigma-Aldrich, estradiol valerate from Aborihan (Iran), naloxone from Tolidaru
(Iran) and oxytocin from Weimer Pharma (Germany). The mean?SEM of contraction
forces (g/100mg tissue) were calculated for each group. The extract was applied
to tissue 2-3min after or before applying stimulants and then, the spasmolytic
effect was calculated after 3min. The results were statistically analyzed with t-test
and ANOVA and P values less than 0.05 were considered as significant.
The (n) represents the number of animals used in each protocol.

Results
and Discussion

Effect
of extract on KCl-induced contraction

KCl (60mM) induced uterus
contraction and after 2min and during the plateau, the extract was added at
0.125, 0.25, 0.5, 1, 2 and 4mg/ml non-cumulatively to the organ bath with 10
min intervals and several refreshing bath solution. As shown in figure 1, the
KCl-induced contraction forces in the several stages of this protocol are not
significantly different. The KCl-induced contractions however, are reduced by
extract in a dose-dependent manner (ANOVA, P<0.0001, n= 9-12). As shown in
figure 2 the extract had the same spasmolytic effect if it was applied before
adding KCl to the tissue (n=8-10).

Effect
of propranolol on spasmolytic effect of extract

Firstly, the
extract (2mg/ml) was applied to the precontracted uterus induced by KCl (60
mM). After 10min and refreshing the bath solution, the same protocol was
carried out in the presence of propranolol (1μM, 1min). As shown in figure
3, in both conditions, the extract induced the spasmolytic effect but this
activity was unaffected by propranolol (n=9).

Effect
of extract on barium chloride-induced contraction

Applying BaCl2 (4mM) to the organ bath
induced the sustain contraction in rat uterus and the extract (2mg/ml) reduced
this contraction (P<0.01, n=6). Moreover, applying extract (2mg/ml) before
adding BaCl2 also reduced the uterus contraction as presented in
figure 4. However, the Ba2+-induced contraction in presence of
extract (2 min) is less than the inhibitory effect of extract on Ba2+-induced
contraction (P<0.05, n=6).

Effect
of extract on oxytocin-induced uterus contraction and the role of calcium

a)??? In normal
calcium De Jalon solution:

To show the contractile
response of uterus to oxytocin, this hormone (10mU/ml) was added to the bath
for 3min and then the tissue was washed. In next stage, extract (0.25mg/ml) was
added for 3min and then in the presence of extract, oxytocin was applied to the
bath for more 3min. This protocol was repeated for 0.5 and 1mg/ml of extract
with 15 min interval and tissue washing. Figure 5 demonstrates that in the
presence of normal calcium solution, the contractions induced by oxytocin were
reduced by the extract in a dose dependent fashion (ANOVA, P<0.0001, n=8).

b)??? In Ca2+-free
De Jalon solution:

This protocol
was the same as previous one, except that, the De Jalon solution was prepared
without calcium. Figure 5 also shows that in the absence of both calcium and
extract, oxytocin induces contraction, which is not as potent as contractions
in the presence of calcium. Extract, however, reduces the oxytocin contractions
dose-dependently (ANOVA, P<0.001, n=6). Statistical analysis indicates that
the extract dose responses in these two conditions are significantly different
(P<0.05) except for 0.5mg/ml extract concentration. The comparison of
reproducibility of oxytocin-induced contractions before applying extract
revealed that the contractions in the presence of calcium are more sustained
than in the absence of calcium (results are not shown).

?

Inhibitory
effect of extract in the presence of naloxone

Effect of the
extract on the KCl-induced uterus contraction was investigated in the absence
and in the presence of naloxone as a non-selective opioid receptor antagonist.
KCl (60 mM) induced contraction was reduced by the extract (2mg/ml). After at
least 15min recovery period and several tissue washing, the same protocol for
KCl and extract was carried out but in the presence of naloxone (1μM,
5min). As demonstvated in figure 6, the spasmolytic effect of extract is not
affected by naloxone (n=7-9).

Study
of anticholinergic activity of extract

Uterus
contraction induced by acetylcholine (0.5μM) was superimposed on the
contraction induced by KCl (30mM) and then atropine (0.5μM) was added to
the bath. As shown in figure 7, the uterus contraction was reduced
(P<0.0001, n=7). Bath solution was exchanged several times and the tissue
left to rest for 30min. Then, atropine (0.5μM) was applied to the bath for
5min and in the presence of atropine, KCl and acetylcholine were added with the
previous concentrations. The figure 7 shows that the uterus responded to the
KCl but not to the acetylcholine. Continuing this procedure (2mg/ml) extract reduced
the uterus contraction (P<0.0001). The inhibitory effect of extract is more
potent than the atropine effect (P<0.01).

This study
demonstrated that Zataria multiflora Boiss hydroalcoholic leaf extract
(ZHLE) can induce spasmolytic effect on KCl-, BaCl2 - and
oxytocin-induced contractions in rat uterus. Although, the antifungal (4-6),
antimicrobial (7), antinociceptive (1), anti-inflammatory (3) and antispasmodic
effects on ZHLE on rat ileum (8) have already been reported but its spasmolytic
activity on uterus is poor investigated. However, the spasmolytic effect of Thymus
vulgaris from the same family (Labiatae) on guinea pig trachea and ileum
were reported (11-12). Extracellular high potassium is well known as smooth
muscle stimulant and a calcium channels (VDCCs) opener (13). Since, verapamile
(L-type VDCCs blocker) deminished the high K+ -induced contraction
in smooth muscle, it is therefore suggested that those substances that decrease
the K+-induced contraction can also block the VDCCs (14). The existence
of L-type VDCCs in rat uterus is also documented (15). Our results indicated
that ZHLE inhibited the KCl-induced uterus contraction. This part of results is
in accordance to spasmolytic effect of ZHLE on rat ileum (8). It has already
been shown that β-adrenoceptors activation in uterus causes relaxation
(16, 17) but the ineffectiveness of propranolol on the extract relaxatory
effect suggested that β-adrenoceptors are not involved in ileum study (8).
Another study showed that barium chloride could induce smooth muscle
contraction by blocking the potassium channels followed by depolarization and
contraction (18) although, it has also been reported that Ba2+
promotes Ca2+ release from intracellular pools in uterus (19) and
stomach (20) smooth muscles. Our results showed that ZHLE reduced the Ba2+-
induced contraction which, results from prevention either calcium efflux or
calcium release but, as mentioned above, the effect of ZHLE should be on the
surface of the cells since this effect was disappeared by washing the tissue as
is discussed in the following.

In next part of
the study, the effect of ZHLE on oxytocin-induced uterus contraction was
investigated in De Jalon solution with low calcium concentration which prevents
the spontaneous motility in uterus and superimposing these contractions with
oxytocin-induced contraction (21). Oxytocin binds to its receptors and
increases inositol triphosphate (IP3) production followed by
promoting calcium release from intracellular pools (22, 23). Therefore,
oxytocin may contract the uterus in Ca2+-free solution (24).
Oxytocin also activates the L-type VDCCs (25). In the present study we
demonstrated that ZHLE reduced the oxytocin-induced contractions in De Jalon
solution with and without calcium. From first part of the obtained results, it
may suggest that ZHLE blocks the L-type VDCCs and therefore relaxes the
contracted uterus. On the other hand, in Ca2+- free solution it
maybe suggested that extract interacted with oxytocin receptors and/or
prevented the calcium release from intracellular pools. In these three main
procedures (uterus contraction by K+, Ba2+ and oxytocin)
the voltage sensitive calcium channels are involved as these contraction were
reduced by the extract. Therefore, it may postulated that ZHLE acts through
blockade of these channels.

It has already been reported that naloxone
antagonized the antinociceptive effect of Zataria multiflora (1), but
the present study showed that the ZHLE spasmolytic effect was unaffected by
naloxone which may suggest that the opioid receptors are not involved in this
activity. Whether the extract has any anticholinergic activity or not, our
results showed that atropine (as a non-selective muscarinic receptor
antagonist) reduced the superimposed contractions of KCl and ACh. In the
presence of atropine, only KCl but not Ach could induce contraction. The
inhibitory effect of the same extract on ACh-induced contraction in rat ileum
has also been reported (8). From this results we concluded that in ZHLE there
is no anticholinergic substance(s) and the inhibitory effect of extract is due
to the action on the common pathway that ACh and KCl cause the contraction.
This common path way is the Ca2+ influx from extracellular fluid.
This part of study also proved the suggested mechanistic effect of the extract
on K+-induced contraction through blockade of VDCCs. Therefore, the
inhibition of the ACh-induced contraction in rat ileum by the extract (8) may
indicate the ACh related consequences of the later events rather than the
involvement of the extract anti-muscarinic property.

In the all procedures (except for oxytocin effect
in Ca2+-free solution) the spasmolytic effect of extract was
reversible, becouse the induced relaxation was disappeared by washing the
affected tissue. We may firelly conclude that, the extract effective
substance(s) induce their effect on the surface of the smooth muscle cells.

Our results
also demonstrated the involvement of calcium channels in spasmolytic effect of Zataria
multiflora Boiss in the rat uterus, which may support its use in folk
medicine to relief dysmenorrhea.?

Acknowledgment

Authors would like to
thank Jondishapur University of Medical Sciences for the financial support.